Seasonal variations of phenolic compounds and biological properties in sage (Salvia officinalis L.)

The aim was to investigate the phenolic content, antioxidant capacity, and antibacterial activity of Dalmatian sage (Salvia officinalis L.) leaves collected during different vegetation periods. Separation and quantification of the individual phenols were performed by reversed-phase (RP)-HPLC coupled with a PDA (photodiode array) detector and using an internal standard, while the contents of total phenols, flavonoids, flavones, and flavonols were determined spectrophotometrically. The antioxidant properties of the sage leaf extracts were evaluated using five different antioxidant assays (FRAP, DPPH, ABTS, Briggs-Rauscher reaction, and β-carotene bleaching). The antimicrobial activity of the extracts was tested against two Gram-positive (Bacillus cereus and Staphylococcus aureus) and two Gram-negative (Salmonella Infantis and Escherichia coli) bacterial reference strains. All extracts were extremely rich in phenolic compounds, and provided good antioxidant and antibacterial properties, but the phenophase in which the leaves were collected affected the phenolic composition of the sage extracts and consequently their biological activity. The May Extract, the richest in total flavonoids, showed the best antioxidant properties and the highest antimicrobial activity. Thus, collection of the plants during May seems the best choice for further use of them in the pharmaceutical and food industry.     

Morphological and proteomic analyses of sugar beet cultures and identifying putative markers for cell differentiation

Normal (N), habituated nonorganogenic (HNO), and tumour (T) sugar beet cell lines were analysed in order to establish specific patterns of extracellular proteins and identify protein markers that might explain the distinct phenotypical characteristics. Electron microscopy showed that the ultrastructure of N cells corresponds to that of parenchyma cells, and that these cells contain plastids with large starch grains. HNO and T cells had enlarged, lobed nuclei with an increased number of nucleoli; the number of nuclei in HNO cells was greater than in T cells. The T plastids were elongated, with reduced thylakoids and abundant phytoferritin deposits, while HNO plastids were small and vacuolated, with an irregular, underdeveloped thylakoid system. The extracellular proteome of the cells was separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Greater differences in protein expression were observed between the HNO and N lines than between the T and N lines. Sixteen of the most prominent bands differentially expressed among the cell lines were cut out from the gel and analyzed by mass spectrometry. Cell wall-modifying enzymes were identified, including a peroxidase whose expression was twofold higher in N and T tissue than in HNO tissue; pectinesterase, which was expressed at a level threefold lower in the T line than in the other cell lines; and xyloglucan endotransglucosylase, which was expressed at a level sixfold higher in HNO and T tissue. Three proteins belonged to the chitinase gene family and their expression was higher in HNO and T tissue than in N tissue. The differential expression of these proteins suggests that these play a role in cell line-specific cell wall composition and cell-to-cell adhesion.     

Fatty acid and phytoplankton compositions of different types of mucilaginous aggregates in thenorthern Adriatic

The biomarkers (fatty acid proportions and ratios characteristicof phytoplankton and bacteria) and phytoplankton species insmall (0.5–2.0 cm) and large (0.5–5 m) marine aggregateswere determined in samples collected in the northern AdriaticSea, during a mucilage event in 1997, as well as in 1993, 1994and 1998, when events were not observed. Types of aggregateswere identified according to various biomarker relationships,particularly those related to bacterial and phytoplankton activitiesand changes in the diatom species composition. Aged mucilaginousaggregates (in summer 1997) showed fatty acid proportions (16P/18P,3.9–7.7) characteristic of the highest phytoplankton activities,and also showed the highest bacterial fatty acid proportions(13.3–17.1%) and ratios (C15:br/C15:0, 4.4–6.0).They showed an different diatom community (dominated by Cylindrothecaclosterium) from that in surrounding waters. These characteristicssuggest a continuous renewal of the aggregate organic matter,supporting the hypothesis that aggregate is a ‘selfsustaining’community. In contrast, both freshly formed marine snow, dominantduring periods without mucilage events, and freshly formed mucilaginousaggregates presented biomarker proportions and ratios similarto those of suspended matter (bacterial fatty acids 3–6.7%,C15:br/C15:0 1.2–4.6, 16P/180.4–4.0), and diatomcomposition similar to that of the ambient water. This indicatesthat marine snow sinks more rapidly than large aggregates, beforesignificant changes can occur in its plankton composition. Otheraging aggregate types showed intermediate characteristics, suggestingthat their residence times in the water column were still sufficientto develop organic production–decomposition cycles thatmodified to various extents their biochemical composition.     

<Emphasis Type="Italic">In vitro</Emphasis> stem elongation of stemless carline thistle

In vitro culture of stemless carline thistle was established using immature zygotic embryos. A satisfactory bud multiplication was achieved on MS medium supplemented with BAP (1 mg L^–1) and IAA (0.2 mg L^–1). Maximum rooting of buds was induced upon short cultivation (4 or 8 days) on an auxin-supplemented medium. Highest number of roots was obtained with NAA in the medium while the longest roots developed on the IAA-supplemented medium. Plantlets that subsequently developed were in rosette form if grown in light (16/8 h light to darkness photoperiod) and with elongated stems if raised in darkness. Light grown plantlets treated with GA₃ showed dose dependent stem increase in length, reaching maximum at the concentration of 10^–4 M. This was correlated with the length and the average number of internodes. If cultivated in the presence of ancymidol, dark grown plantlets showed reduced stem length. However, the inhibitory effect of the growth retardant on stem elongation was completely overcome by the addition of GA₃.     

Spectrum of cystic fibrosis mutations in Serbia and Montenegro and strategy for prenatal diagnosis

We have screened 175 patients for molecular defects in the cystic fibrosis transmembrane conductance regulator (CFTR) gene using nondenaturing polyacrylamide gel electrophoresis (PAGE), denaturing gradient gel electrophoresis (DGGE), and sequencing. Six different mutations (F508del, G542X, 621+1G --> T, 2789+5G --> A, R1070Q, and S466X) accounted for 79.71% of CF alleles, with the F508del mutation showing a frequency of 72.28%. Another 12 mutations (R334W, 2184insA, I507del, 1525-1G --> A, E585X, R75X, M1I, 457TAT --> G, 574delA, 2723delTT, A120T, and 2907delTT) covered an additional 3.36%. A novel mutation (2723delTT) was found in one CF patient (F508del/2723delTT). Thus, a total of 18 mutations cover 82.57% of CF alleles. During our study, 72% of families at risk for having a CF child were found to be fully informative for prenatal diagnosis. Prenatal diagnosis was performed on 56 families; 76 analyses resulting in 16 affected, 38 carriers, and 22 healthy fetuses. These results imply that the molecular basis of CF in Serbia and Montenegro is highly heterogeneous, as is observed in other eastern and southern European populations. Because we detected more then 80% of CFTR alleles, results could be used for planning future screening and appropriate genetic counseling programs in our country.     

Regulation of corticotropin-releasing hormone receptor type 1alpha signaling: structural determinants for G protein-coupled receptor kinase-mediated phosphorylation and agonist-mediated desensitization

Attenuation of CRH receptor type 1 (CRH-R1) signaling activity might involve desensitization and uncoupling of CRH-R1 from intracellular effectors. We investigated the desensitization of native CRH-R in human myometrial cells from pregnant women and recombinant CRH-R1alpha stably overexpressed in human embryonic kidney (HEK) 293 cells. In both cell types, CRH-R1-mediated adenylyl cyclase activation was susceptible to homologous desensitization induced by pretreatment with high concentrations of CRH. Time course studies showed half-maximal desensitization occurring after approximately 40 min of pretreatment and full recovery of CRH-R1alpha functional response within 2 h of removal of CRH pretreatment. In HEK 293 cells, desensitization of CRH-R1alpha was associated with receptor phosphorylation and subsequent endocytosis. To analyze the mechanism leading to CRH-R1alpha desensitization, we overexpressed a truncated beta-arrestin (319-418) and performed coimmunoprecipitation and G protein-coupled receptor kinase (GRK) translocation studies. We found that GRK3 and GRK6 are the main isoforms that interact with CRH-R1alpha, and that recruitment of GRK3 requires Gbetagamma-subunits as well as beta-arrestin. Site-directed mutagenesis of Ser and Thr residues in the CRH-R1alpha C terminus, identified Thr399 as important for GRK-induced receptor phosphorylation and desensitization.We conclude that homologous desensitization of CRH-R1alpha involves the coordinated action of multiple GRK isoforms, Gbeta gamma dimers and beta-arrestin. Based on our identification of key amino acid(s) for GRK-dependent phosphorylation, we demonstrate the importance of the CRH-R1alpha carboxyl tail for regulation of receptor activity.     

Immunohistochemical appearance of HNE-protein conjugates in human astrocytomas

Gliomas are tumors originating from astrocytes, oligodendrocytes or ependimal cells. Those of astrocytic origin are the most widespread of primary brain tumors and account for more then 60% of all CNS neoplasms. The current state of knowledge on the associations between tumor etiology and oxidative stress suggests that environmental factors that cause oxidative stress could also induce and promote cancer, especially in case of hereditary predisposition. Among mediators of oxidative stress, lipid peroxidation product 4-hydroxynonenal (HNE) is of particular relevance in oncology, as it is known to act as a growth-regulating factor and a signaling molecule. The aim of present study was to investigate by immunohistochemistry the presence of HNE-modified proteins in different types of astrocytoma. Our study comprised 45 astrocytic tumors. These tumors were graded in accordance with the WHO classification as diffuse astrocytomas (DA), anaplastic astrocytomas (AA) and glioblastomas (GB), while each group comprised 15 tumors. Slides of paraffin-embedded tumor tissue were stained with hematoxylin-eosin or were prepared for immunohistochemistry with monoclonal antibodies to HNE-histidine conjugate. Positive immunohistochemical reaction to HNE was analyzed semi-quantitatively. HNE positivity was proportional with malignancy of astrocytomas. The weakest presence of HNE-histidine adducts was found in DA, followed by AA and GB. Lowest intensity of HNE immunopositivity was present in tumor cells of almost all DA, predominantly around blood vessels. In malignant variants of astrocytoma, AA and GB, HNE positivity was moderate to strong, and diffusely distributed in all tumors.     

Biomembranes from slaughterhouse blood erythrocytes as prolonged release systems for dexamethasone sodium phosphate

The present study investigated preparation of bovine and porcine erythrocyte membranes from slaughterhouse blood as bio‐derived materials for delivery of dexamethasone‐sodium phosphate (DexP). The obtained biomembranes, i.e., ghosts were characterized in vitro in terms of morphological properties, loading parameters, and release behavior. For the last two, an UHPLC/–HESI–MS/MS based analytical procedure for absolute drug identification and quantification was developed. The results revealed that loading of DexP into both type of ghosts was directly proportional to the increase of drug concentration in the incubation medium, while incubation at 37°C had statistically significant effect on loaded amount of DexP (P < 0.05). The encapsulation efficiency was about fivefold higher in porcine compared to bovine ghosts. Insight into ghosts’ surface morphology by field emission‐scanning electron microscopy and atomic force microscopy confirmed that besides inevitable effects of osmosis, DexP inclusion itself had no observable additional effect on the morphology of the ghosts carriers. DexP release profiles were dependent on erythrocyte ghost type and amount of residual hemoglobin. However, sustained DexP release was achieved and shown over 3 days from porcine ghosts and 5 days from bovine erythrocyte ghosts. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1046–1055, 2016     

Inhibition of CDK-mediated Smad3 phosphorylation reduces the Pin1-Smad3 interaction and aggressiveness of triple negative breast cancer cells

Triple negative breast cancer (TNBC) is a highly aggressive breast cancer subtype that lacks effective targeted therapies. Although TNBC is not defined by specific therapeutic targets, a subset of patients have tumors that overexpress cyclins. High cyclin D/E expression catalyzes CDK4/2 activity. In turn, CDK4/2 can non-canonically phosphorylate Smad3, a key TGFβ signaling intermediate, and this phosphorylation has been associated with the shift from tumor-suppressive to oncogenic TGFβ pathway action in breast oncogenesis. Additionally, CDK-mediated Smad3 phosphorylation facilitates an interaction between Smad3 and Pin1, a cis-trans isomerase that is also overexpressed in aggressive breast cancers. Treatment with CYC065, a CDK2/9 inhibitor, decreased non-canonical Smad3 phosphorylation and inhibited the Pin1-Smad3 interaction. We hypothesized that the interaction of Pin1 and Smad3, facilitated by CDK-mediated Smad3 phosphorylation, promotes TNBC cell aggressiveness. Inhibition of the Pin1-Smad3 interaction in TNBC cell lines, through depletion of Pin1 or CYC065 treatment, resulted in decreased cell migration/invasion and impeded the EMT program. Inhibition of CDK-mediated phosphorylation of Smad3 by mutagenesis also decreased cell migration, underscoring the importance of non-canonical CDK2 phosphorylation of Smad3 to enable cell motility. Pin1 depletion restored Smad3 protein levels and tumor-suppressive activity, suggesting that the Pin1-Smad3 interaction has a negative impact on canonical Smad3 action. Collectively, the data show that the Pin1-Smad3 interaction, facilitated by CDK-mediated Smad3 phosphorylation, is associated with oncogenic TGFβ signaling and breast cancer progression. Inhibition of this interaction with CYC065 treatment may provide an important therapeutic option for TNBC patients.